RCA is employed in the replication of circularized DNA sequences. RCA products are used in DNA sequencing, cloning, library construction, probe generation and genetic screening. More recently, RCA products are employed in cellular expression, wherein cells are administered with naked DNA to produce an RNA or protein; and in DNA vaccination, wherein an organism is administered with naked DNA to produce an immunological response. In the case of DNA vaccination, rather than administering the pathogen itself (generally in a dead or disabled form so as to minimize any risk of actual infection), as in standard vaccinations, some portion of the pathogen's genome is administered and is then expressed by the organism in a manner sufficient to elicit an immune response. Because only a portion of the pathogen's genome is administered, there is little or no risk of actual pathogenic infection.
To date, DNA for cell transfection and DNA vaccines is produced using plasmid DNA. However, the use of plasmid DNA requires labor-intensive and expensive plasmid purification and increases the risk of contamination by extraneous bacterial components such as proteins, DNA, RNA, small molecules or purification reagents (e.g., ethidium bromide, chloroform, phenol, etc.). Any of these contaminants may have undesirable consequences.
Methods developed more recently employ cell-free RCA techniques. These techniques do not require plasmid purification and therefore reduce the risk of contamination, and are better suited for expression in cellular systems and therapeutic applications, such as DNA vaccines. However, to ensure sufficient uptake and expression in the DNA recipient, such techniques have, to date, required extensive post-amplification processing of the RCA product, wherein the product is broken into shorter units (monomers, dimers, trimers, etc.) and then circularized or supercoiled. Such processing is expensive and time intensive.